Guiding role of esophageal variceal diameter in treatment of endoscopic ligation: an animal experimental study

In this study, according to the Location, Diameter, Risk factor (LDRf) classification principle, the influence and effect of esophageal varices diameter on the degree of complete ligation of multicyclic ligator were investigated. Methods. The esophageal veins of healthy piglets were filled with methylene blue solution, and the in vitro pig esophageal varices model was made, which were divided into three groups according to the diameter of esophageal varices: D1, 0.4–1.0 cm; D2, 1.1–1.5 cm; and D3, 1.6–2.0 cm. Finally, the ligation effect of each group was analyzed statistically. A total of 407 ligations were performed on the simulated esophageal variceal model. There were 103 ligations in the D1 group and 98 were complete (95.15%, 98/103); 151 ligations in the D2 group and 47 were complete (31.13%, 47/151); and 153 ligations in the D3 group but none were complete (0%, 0/153). There was significant difference in the degree of complete ligation between the two groups (χ2 = 38.0014, P ≤ 0.001). In the varicose ligation model, the complete ligation effect was the most complete and robust when the varicose diameter was 0.4–1.0 cm. This study showed that the varicose vein diameter in LDRf classification was reasonable and feasible to guide endoscopic varicose vein ligation.


Materials and methods
We made model EVL of animals pig, and this simulated experimental study was carried out according to the LDRf classification criteria 15 .Institutional Animal Care and Use Committee of Capital Medical University (protocol number: 201203031-001) and PLA General Hospital (protocol number: 20110323-001) approved all procedures.All experimental procedures were performed in accordance with relevant guidelines and regulations.This study is reported in accordance with ARRIVE guidelines, https:// arriv eguid elines.org.

Experimental animals
Experimental pigs, 3-24 months old, all female, weighing 40 ± 20 kg, were clinically healthy and were purchased from Liuli River Experimental Pig Farm, Fangshan District, Beijing.

Experimental equipment and drugs
Experimental equipment includes anesthesia machine, monitor, anesthesia bed, laryngoscope, stethoscope and other commonly used items.The equipment includes Olympus-240 electronic gastroscope, Wilson-Cook sixshot ligation device, Vernier caliper, and T-shape tubes.Glucose methylene blue solution was used to fill the veins for visibility.

Anesthesia method
Piglets were given intravenous general anesthesia, and the anesthesia method is divided into two stages:

Anesthesia induction phase
Administration before induction: Intramuscular injection of atropine 0.5 mg and dexamethasone 10 mg is routinely given before anesthesia induction to reduce the secretion of salivary glands and tracheal glands and prevent allergic reactions.The anesthesia induction drugs were midazolam 0.1-0.2mg/kg and Sumisan 0.25-0.3ml/kg compound drugs.

Anesthesia maintenance phase
The anesthesia maintenance medication is to use 6 succinylcholine, 6 diazepam injection, and 7 lidocaine to be added to 500 ml of balanced solution to prepare an anesthetic mixture for slow intravenous infusion, first rapid infusion of 5-10 ml, and after the experimental animal anesthesia is complete, the drip rate is slowed down to 40-60 drops/min.

Selecting the pig veins
We opened the abdominal cavity of the pigs and selected the inferior vena cava, portal vein, superior mesenteric vein, and limb veins of different thickness.

Pig variceal model and measurement of varicose vein diameter
One end of the vein was ligated and the other end connected to the T tube, whose other end was connected to the glass column burette filled with methylene blue liquid The T tube was opened so that the water flowed into the vein, and the water column gradually decreased, while the porcine vein was gradually filled.When the water flow stopped, the end of the vein that was connected to the T tube was ligated to produce porcine variceal models with different diameters ranging from 0.4 to 2.0 cm and lengths of 3.0-4.0cm, as measured with a vernier caliper (Figs. 1, 2).

Selecting the pig esophagus
The pig's esophagus is about 40 cm long, divided into three sections, each about 13 cm long,, and the esophageal lining was flipped out.The mucosa and muscularis propria layers were bluntly dissected with a hemostat, forming a submucosal tunnel (Figs. 3, 4).

Pig model of esophageal varices
Using a hemostat, the pig modeled varicose vein was introduced through the esophageal tunnel between the muscularis propria and submucosal layers, to form the pig model of esophageal varices.

Measuring esophageal variceal diameter
The diameter of the esophageal varices was measured by a vernier caliper, and divided into three groups according to the LDRf classification of the variceal different diameters: D 1 , 0.4-1.0cm; D 2 , 1.0-1.5 cm; and D 3 , 1.6-2.0cm (Fig. 5).

Pig variceal ligation model
The multiple-shot ligation device pre-fixed to the front end of the endoscope was used to target the esophageal varicose vein to create continuous suction.When the suction pressure rose to 0.03-0.05MPa 18 and the endoscopic vision gradually became completely blue, the rubber band was released to stop the suction and ligated at the base of the vein 19 (Fig. 6).The esophageal mucosa was cut off and separated after ligation, and the vein was dissected after submucosal ligation.The ligation effect was observed and judged as complete (100%), incomplete (50%), and no ligation (Fig. 7a-c).

Statistical analysis
We have used SPSS Version 13.0 software to carry out the statistical analysis which is given in this manuscript.P < 0.05 was considered statistically significant.

Esophageal variceal diameter
A total of 407 ligation models of simulated esophageal varices were formed: 103 in the D 1 group, 151 in the D 2 group, and 153 in the D 3 group.

Discussion
Endoscopic treatment of esophageal varices is mainly by ligation and supplemented by sclerosis.. Ligation and sclerotherapy have their own advantages and disadvantages, and should be based on the characteristics of each method.Re-examination of gastroscopy 1 week after emergency ligation of severe esophagogastric varices and the discharge time should be determined according to the results of gastroscopy, which can effectively reduce rebleeding after banding.Severe esophageal varices should be mainly ligation, with emphasis on preventing  www.nature.com/scientificreports/re-bleeding and alleviating varices.Mild and moderate esophageal varices should be mainly scleroded, focusing on the treatment of deep mucosal varices and perforating branches to reduce recurrence, and at the same time, due to the small amount of sclerosing agent, the incidence of complications after sclerosing agent injection is effectively reduced.When necessary, the treatment should be supplemented with transparent caps and tissue glue.EVL therapy is one of the latest methods in treatment of esophageal variceal bleeding, which was first reported by Stiegmann in 1986 20 .The therapeutic effect of EVL has the following mechanisms 21 : (1) mechanical blockade of the blood flow of the varicose veins to make the varices shrink; (2) thrombosis at the ligation site of the varicose vein and occurrence of organizing or calcification; (3) scar formation and fibrosis in the wall of the ligated vein; (4) degradation or disappearance of the varicose veins after ligation.
Esophageal variceal bleeding is the major complication and cause of death in patients with cirrhosis.How to seize the opportunity to reduce or eliminate the esophageal varicosis is the most important means to prevent bleeding.
The principle of EVL is to block the varicose vein blood flow emergency hemostasis, so that the ligation of venous thrombosis, tissue necrosis, fibrosis formation, and finally make the varicose vein disappear.EVL includes single ligature, multiple ligature and nylon rope ligature.Repeated ligation is superior/not inferior to repeated sclerotherapy in the treatment of esophageal varices, but the incidence of side effects (including esophageal stenosis, perforation, infection, etc.) is significantly lower.Simultaneous combination of ligation and sclerotherapy for esophageal variceal vein rupture bleeding is not superior to repeated ligation treatment [22][23][24][25] .
Many studies have shown 90% hemostasis after emergency therapy for esophageal variceal bleeding by endoscopic ligation 26 .Ligation therapy has become the first choice of endoscopic treatment with good results, due to the rapid disappearance of varices, few complications, simple operation, and low rebleeding rate after ligation.However, variceal ligation has some risk, especially the ligation ring may fall off triggering the bleeding which is difficult to control.Three results can occur after ligation of esophageal varices: (1) complete ligation, indicating that the ligation effect is certain and complete, and the ligation ring at the root of the ligated varicose veins is not easy to slip off and is strong and stable; (2) incomplete ligation (the root of the varicose veins is not completely ligated, ligation only less than or equal to 50%), indicating that the effect of banding was uncertain and incomplete, and the ligature knot was easy to fall off early, which was easy to induce massive bleeding and increase mortality.(3) ligation failure, indicating that the banding ring cannot ligature the root of varicose veins.
From above consideration, it is necessary to carry out clinical studies on the surgical methods, indications and other aspects of EVL.Studies have shown that the factors affecting EVL efficacy include the following.(1) Variceal diameter > 1.0 cm, at which point the ligature ring is unable to tie the entire varicose vein, and the ligature ring often falls off, resulting in fatal bleeding.(2) Ligation techniques that directly affect the efficacy include: correct positioning of varicose veins, adequate attachment of sucking, making the venous bulb in the transparent cap as full as possible, and avoiding ineffective sucking.This study shows that for varicose vein diameter > 1.5 cm, not completely ligation.For varicose vein diameter < 0.3 cm, the ligation device directly inhaled the esophageal muscle layer into the transparent cap; Then ligation the muscle layer.In this case, the patient experienced longterm postoperative pain and scar stenosis.(3) For varicose veins with severe mucosal erosions or blood blisters, excessive sucking during ligation can sometimes cause severe mucosal damage and induce bleeding, which directly affects the efficacy 27 .
In this study, in the 103 variceal ligations in D1 group (diameter 0.4-1.0cm), the rate of complete ligation was 95.15% (98/103), it indicates that the ligated vascular root is firm and determined.In 151 variceal ligations in D 2 group (diameter 1.1-1.5 cm), the rate of complete ligation was 31.13%(47/151).However, in 153 variceal ligations in D 3 group (diameter 1.6-2.0cm), the rate of complete ligation was 0%.Thus, for varices with diameter 0.4-1.0cm, the ligation effect was most complete, and the success rate was the highest, while the ligation failure rate was highest for varices > 1.6 cm in diameter.Therefore, it is particularly important to measure the diameter www.nature.com/scientificreports/ of esophageal varices on an endoscopic scale 27,28 .This can better apply LDRf typing to guide endoscopic therapy, reduce the risk of endoscopic variceal hemorrhage, and improve the success rate of hemostasis.In this study, the ligation diameter of esophageal varices in pigs was < 1.5 cm, and this experimental results are consistent with those reported in previous literatures.EVL has rapid eradication of varicose veins and few complications, but the recurrence rate of varicose veins is high.EVL can block the bleeding collateral of the left gastric vein, esophageal vein and vena cava, but after the blood flow of the esophageal vein is blocked, the gastric coronary vein and the perigastric venous plexus have vascular dilation, increased blood flow, and increased recurrence rate over time, so repeated ligation and consolidation therapy are often required [21][22][23][24] .
When cirrhosis portal hypertension occurs, the pressure in esophageal varices increases, causing venous dilation, diameter increase, venous wall thinning, and venous wall tension increase.When the critical value is reached, the venous wall rupture bleeding [29][30][31] .Our previous experimental studies have confirmed that the esophageal variceal pressure is a major factor affecting ligation 32 .
To date, there have been published literature on life threatening massive bleeding due to ulceration following variceal banding and early spontaneous slippage of rubber bands [33][34][35] , especially 5 to 10 days after the procedure when the O-ring detaches.However, we will be unable to predict the presence of an ulceration with a residual vein at its base and to evaluate the thrombosis of that vein after EVL until detachment of the O-ring 36 .There are other literature reports that percutaneous transhepatic obliteration (Percutaneous transhepatic obliteration, PTO) is one of the useful rescue procedures for life-threatening bleeding after EVL for severe esophagogastric varices 37 .or additional sclerotherapy immediately after EVL may be required to prevent this fatal complication.Thus ,further studies will be needed in order to identify the specific risk factors for potentially fatal complications of this type 38 .
In this experimental model, one limitation of the present study was that we used normal vessels dilated that do not correspond to the natural esophageal varices formation since the varicose veins vessels are not normal because their varicose veins angiogenesis happen as a vessel slowly becomes blocked, and the lack of the present method is that could not be compared to the natural esophageal varices produced by cirrhosis.I think although interfering in the results for guiding role of esophageal variceal diameter in treatment of endoscopic ligation, but this experiment is based on the previous research project "Esophageal variceal pressure influence on the effect of ligation" 31 , and variceal veins of different diameters were simulated with the esophagus of animal pigs to perform endoscopic ligation experiments, and further verified the guiding effect of variceal vein diameter on endoscopic ligation in LDRf typing.So, I believe this animal simulation experiment has preliminarily verified the guiding effect of varicose diameter in LDRf typing on endoscopic ligation.However, a large number of clinical studies are needed to verify the guiding role of LDRf typing.Certainly, the results of this animal simulation experiment maybe it would be more efficient as a simulation for endoscopic ligation training.
Therefore, multi-factor experiments are needed, and measuring the diameter and pressure of esophageal varices has important clinical significance for evaluating the risk of bleeding and its control effect.

Figure 5 .
Figure 5.In vitro measurment of the diameter of the esophageal varices.

Figure 6 .
Figure 6.In vitro ligation of the esophageal varices.

Figure 7 .
Figure 7.The effect of ligation pattern.

Table 2 .
According to LDRf type, selection of treatment options based on the diameter grading and risk Presence of fresh blood, excluding non-variceal bleeding